Japan has reached a society composed largely of elderly people and the average life span is highest in the world. The hope of people is attaching importance to the quality of life (QOL) rather than to mere life prolongation, and speaking and eating are important functions to lead to the be-all of particularly the elderly people's existence and in that sense, health maintenance of organs of mastication including the preservation of teeth can be said an important factor in controlling QOL. Mastication is an inevitable function of ingestion, and furthermore according to the recent study of the system of mastication, it is being clarified that mastication affects various systemic functions such as urging of development and activation of mind/nerves by stimulating brain cells, enhancement of immune functions and further inhibition of obesity. Accordingly, the decline of the function of mastication due to the loss of teeth leads to a possibility of accelerating the way to dementia and causing life style related diseases or the like. At present, with the middle-aged and the elderly of 35 to 65 ages, the ratio of population affected by periodontitis is more than 80% and the demand of the people is high in treating periodontitis and its contents are varied. Many associated factors complicatedly have a part in the onset and progression of periodontitis, and the preventive treatment centering on the oral cavity cleaning which has been performed heretofore cannot solve periotontitis. People who unfortunately get periodontitis and are treated mostly demand functional recovery and an aesthetic improvement and at this stage, the treating method centering on the non-regenerative treating method which has been performed heretofore for these demands can be said insufficient and a revolutionary treating method which can regenerate lost periodontal tissues is demanded.
Teeth erupt through the epithelium and are exposed into the oral cavity and lose continuity on the boundary between the teeth and the gingiva and are in a very specific environment in a living body. The teeth and the gingival are constituted of “epithelium adherence” and “connective tissue adherence. As to the former, the epithelium called as junctional epithelium adheres to the dental surface (enamel) through the hemidesmosome and the basal plate. The latter is constituted of the periodontal ligament, and while collagen tissues are mineralized in the cementum of the dental root surface, the tissues anchors into the alveolar bone while being also mineralized to migrate into the gingival tissue, and accordingly teeth are firmly bonded to the alveolar bone and the gingiva.
Periodontitis is an inflammatory disease in the periodontal tissue caused by plaque bacteria and classified into “periodontitis” and “gingivitis”. The inflammation limited to the gingiva is called as “gingivitis” and the inflammation leading to the periodontal ligament and the alveolar bone to destruct the adhesion by the periodontal ligament is called as “periodontitis”. In general, the gingivitis proceeds to periodontitis to result in the formation of a pocket (a groove) in the periphery of teeth. With the pocket deepened, the plaque bacteria in the pocket proliferate to advance the inflammation to the depth. The localized restoring factor as called occlusal traumatism which is found when somatic modified factors (a drug, a blood disease, an immunological disease, nutritional state, stress, fatigue, smoking and the like) are involved and, in addition, the mechanical load to teeth such as grinding of teeth becomes excessive to worsen the inflammation. According to the case of disease seized with periodontitis to highly advance tissue destruction, once lost periodontal tissues cannot be restored in the original form and functions even when tooth extraction is avoided and after treatment, a remarkable functional/aesthetic damage remains to come to a big factor to lower QOL of the patient.
The resorption of the alveolar bone is one of the states of disease characteristic to periodontitis, and the simultaneously occurring loss of the dental root cementum and the periodontal ligament (periodontal ligament) is the essential state of disease. The periodontal ligament indicates a syndesmotic structure as its name shows and suspends teeth in the socket of the alveolar bone through the cementum of the dental root surface to play a role of cushioning a strong bite pressure and is rich in vascular components and has a high metabolic activity to play an important role of maintaining the homeostasis of periodontal tissues. In spite of many studies to aim at the regeneration of periodontal tissues over nearly half a century, an appropriate treatment method has not been established yet because simultaneous regeneration of both the alveolar bone and the periodontal ligament tissue is very difficult. It is clarified that without the regeneration of the periodontal ligament, intrusion of the oral cavity epithelium into a defect and bony ankylosis of the dental root are caused to be healed in a biologically unstable state, and they come to the cause of inviting the recurrence of periodontitis and the deciduation of tooth
In order to treat this periodontitis, scaling/root planing S/RP) is being performed. This is an operation method which comes to the basis of the treatment of periodontitis and comprises mechanically removing infectious tissues at the affected site of periodontitis, mainly removing the cementum of the dental root surface, the periodontal ligament and the gingival tissue which are contaminated by bacterial infection with the use of specific equipment and attaching the peripheral tissue in an easy adherent form to the dental root surface to expect spontaneous cure. The healing form is mainly epithelial adherence formed by the epithelium which is allowed to proceed and proliferate on the dental root surface to be treated. This method does not require a surgical treatment and enables the treatment without anesthesia or by local infiltration anesthesia alone and is a necessary and sufficient treating method if the site to be treated has no aesthetic and functional problem. However, in a case of a destructed alveolar bone in an advanced stage of periodontitis, when reinfection is presumed after treatment, this treating method alone is not sufficient. Further, at the site complicated in the shape of a tooth, this operation method tends to be difficult, and in this case a surgical treating method is applied. The site complicated in the shape of the tooth which cannot be treated by S/RP alone is provided with a surgical treatment which surgically forms a gingival flap to visually treat it. The healing form basically expects the same epithelial adherence as in S/RP.
Under these circumstances, the regenerative medicine technique for positively reconstructing periodontal tissues has been being actively studied. Several techniques are already utilized in the treatment and include, for example, excision of tooth, bone transplantation and mucous ligament transplantation. This alveolar bone transplantation is classified into bone autotransplantation, allotransplantation heterotranplantation and artificial bone transplantation. The bone autotransplantation is mainly performed and is a method of transplanting a bone segment collected from another site in the oral cavity into a bone defect. Further, a decalcified freeze-dried bone allotransplantation (DFDBA) is used in Western countries to get better clinical results. As the artificial bone, hydroxyapatite (HA) is used as a representative. The healing form has an aspect of effectively acting on the retention of blood clot in a defective site compared to S/RP alone, and it is thought that the conditions of inhibiting the intrusion of the epithelium and simultaneously enabling easy migration of the periodontal ligament cells can be provided but it is reported that the main healing form in animal experimentation mainly comes to epithelial adherence which does not cause the regeneration of neocementum and periodontal ligament. The bone autotransplantation has a smallest problem of antigenicity and infection and can be naturally easily accepted in the receptive part. Further, it has an advantage of expecting bone conductivity and bone inductiveness in bone transplant materials. However, it is very difficult to secure a necessary amount of transplant bones in bone autotransplantation. Further, the problem of antigenicty/infection remains in the bone allo- and hetero-transplantation, and there has been a problem that independently of the types of transplant materials, bony ankylosis of a tooth having a large bone defective site and a large area of a transplant bone in contact with the dental root surface is caused.
Between the late 1970's and 1980's, a research group in Scandinavia confirmed that the epithelial tissue and the gingival connective tissue are lacking in the regenerative capacity and the bone tissue causes bone ankylosis on the dental root surface and found that for the regeneration of periodontal ligament tissues, it is necessary for the cells derived from periodontal ligament tissues to be present in a defect, particularly on the dental root surface. It is the “guided tissue regeneration method” (GTR method) that was devised on the basis of this concept. This method enables the regeneration of periodontal ligament tissues by inhibiting the intrusion of the epithelial cell which has a high proliferation speed to early proceed to the defect with a biocompatible shielding ligament to simultaneously perform a space-making in the defect. At present, this method can most expect the regeneration of periodontal tissues and gets better results in the site showing a bone defective form easy in space making. However, this method had problems such that the clinical operation method was complicated; as to the site showing a complicated defective form, it was difficult to accurately place the biocompatible ligament on the dental root surface; the ligament had to be completely covered with a gingival flap during healing due to the standpoint of infection; and prognosis was easily affected by the operation method and the environment. Further, the biocompatible ligament is of absorptiveness and of nonabsorptiveness, and the use of the former required reoperation for removing the ligament and the use of the latter did not require reoperation but a problem of its strength for performing space making remained. Since the cell migration from the residual periodontal ligament is expected, the amount reproduced is limited and additionally, viewed from the retention of the ligament, an applied case is limited to part of the vertical bone defect. In the diffuse chronic periodontitis widely seen in the middle-aged and the elderly, the main state of disease is horizontal bone resorption, and not a few cannot be coped with this GTR method.
Thus, even if periodontal ligament-derived cells are understood to be an indispensable factor to the regeneration of periodontal tissues, in the present situations, the conventional treatment methods including this GTR method only expect the cell migration from the residual tissues in spite of the application of a growth factor and are greatly affected by the defective form and the amount of the residual periodontal tissues to limit its indications. In highly damaged periodontal tissues, mere expectation of the migration of cells of a basis for regeneration from the residual peripheral tissues is not sufficient and the necessity to externally supply periodontal tissues is being clarified. The object of regenerative treatment in periodontal tissues places the focus on how to inhibit the healing by the epithelial adherence to acquire the connective tissue adherence by the periodontal ligament.
In recent years, several studies of aiming at the tissue regeneration by the cell transplantation method are reported. These studies are mostly to perform the injection transplantation of a three-dimensional matrix seeded with a single type of cells to a tissue defect but any of them has not yet realized an expected tissue reconstruction. The reason is thought to be the difficulty in the selection of a cell source and the control of the localization of cell differentiation in the tissue defect. The regeneration of periodontal tissues is inevitably accompanied by cementification on the dental root surface, and it is necessary that both the periodontal ligament of soft tissues and the cementum or the alveolar bone of hard tissues are simultaneously regenerated and functionally connected to each other. When these two types of tissues are formed with a time lag by the action of the precursor cell and the growth factor which are different and tissue-specific, the cell transplanting method in the regeneration of periodontal tissues must be more delicate. In other words, not that a single type of cells is merely injected to the defect undergone space-making and left to tissue differentiation in the living body but that it is necessary to regulate the site of arrangement of cells to arrange the cells at respective appropriate sites.
The cells necessary in this instance have been cultured on the surface of glass or the surface of a synthetic polymer having undergone various treatments. Various vessels made of a material such as polystyrene having undergone surface treatment such as γ-ray irradiation and silicone coating are popularized as cell culture vessels. The cultured/proliferated cells with the use of such cell culture vessels are detached/recovered from the surface of the vessels by treatment with a protease such as trypsin and a chemical. However, such defects have been pointed out that the recovery of the proliferated cell by providing the above described chemical treatment makes the treatment step complicated, has a high possibility of mixing of impurities and sometimes damages the inherent functions of the cells due to the denaturation or the damage of proliferated cells by the chemical treatment.
In order to overcome these defects, several techniques have been proposed. Of them, particularly Japanese Patent Application No. 2001-226141 (Japanese Laid-open Patent Publication No. JP 2003-38170A) has enabled the preparation of a cell sheet having sufficient strength by culturing an anterior eye part-related cell on a cell culture support composed of a base material whose surface is coated with a temperature-responsive polymer having an upper limit or lower limit critical dissolution temperature in water of 0 to 80° C., if necessary or required, making the cultured cell layer a multilayer by the conventional method and detaching the cultured cell sheet only by changing the temperature of the support. Further, this cell sheet maintains a basement ligament-like protein as well and clearly improves bioadherence to tissues compared to the above described dispase-treated cell sheet. Further, according to WO/2002/008387, by culturing cells of the heart muscle tissue on a cell culture support comprising a base material coated with a temperature-responsive polymer to obtain a heart muscle-like cell sheet, thereafter changing the culture fluid temperature to the upper limit critical dissolution temperature or higher or to the lower limit critical dissolution temperature or lower, allowing the multilayered cultured cell sheet to adhere to a polymer film and detaching the cell sheet together with the polymer film, and further making the cell sheet a three-dimensional structure, it was found that a cell sheet having several functions with a reduced structural defect as a heart muscle-like tissue in vitro and a three-dimensional structure could be constructed. However, in both methods, no examination on the regeneration of the periodontal ligament and the acquirement of connective tissue adherence has been made.